The invention relates to a human chemokine receptor, and to compositions and methods useful for diagnosing and treating physiologic and pathologic conditions mediated by the receptor and its ligand. The invention finds application in the biomedical sciences.
Chemokines are a class of cytokines that play important roles in inflammatory responses, leukocyte trafficking, angiogenesis, and other biological processes related to the migration and activation of cells. As mediators of chemotaxis and inflammation, chemokines play roles in pathological conditions. For example, the concentration of the chemokine MCP-1 is higher in the synovial fluid of patients suffering from rheumatoid arthritis than that of patients suffering from other arthritic diseases.
Known chemokines are typically assigned to one of four subfamilies based on the arrangement of cysteine motifs. In the so-called alpha-chemokines, for example, the first two of four cysteines (starting from the amino terminus) are separated by an intervening amino acid (i.e., having the motif C-X-C). The beta-chemokines are characterized by the absence of an intervening amino acid between first two cysteines (i.e., comprising the motif C-C). The smaller gamma- and delta-chemokine families are characterized by a single C residue (gamma) or a pair of cysteines separated by three residues (delta; ie., comprising the motif CX3C). For a recent review on chemokines, see Ward et al., 1998, Immunity 9:1-11 and Baggiolini et al., 1998, Nature 392:565-568, and the references cited therein.
Chemokine activity may be mediated by receptors. For example, several seven-transmembrane-domain G protein-coupled receptors for C-C chemokines have been cloned: a C-C chemokine receptor-1 which recognizes MIP-1xcex1, RANTES, MCP-2, MCP-3, and MIP-5 (Neote et al., 1993, Cell, 72:415-415); CCR2 which is a receptor for MCP1, 2, 3 and 4 or 5; CCR3 which is a receptor for RANTES, MCP-2, 3, 4, MIP-5 and eotaxin; CCR5 which is a receptor for MIP-1xcex1, MIP-1xcex2 and RANTES; CCR4 which is a receptor for MDC or TARC; CCR6 which is a receptor for LARC; and CCR7 which is a receptor for SLC and ELC (MIP-3xcex2; reviewed in Sallusto et al., 1998, Immunol. Today 19:568 and Ward et al., 1998, Immunity 9:1-11).
Due to the importance of chemokines and their receptors as mediators of chemotaxis and inflammation, a need exists for the identification, isolation, and characterization of members of the chemokine receptor family to facilitate modulation of inflammatory and immune responses.
In one aspect, the invention provides a new chemokine receptor, CCX CKR. In one embodiment, the invention provides an isolated, substantially pure, or recombinant CCX CKR polypeptide, or immunogenic fragment thereof. In one embodiment the: polypeptide has the amino acid sequence identical to SEQ ID NO:2. In another embodiment, the polypeptide with an amino acid sequence that differs from SEQ ID NO:2 by conservative mutations, which is at least 60%, 80%, or 90% identical to SEQ ID NO:2, and/or that is immunologically cross-reactive with the full-length polypeptide encoded by SEQ ID NO:2. In one embodiment, the polypeptide of the invention is a fusion protein. In some embodiments, the polypeptide of the invention has an activity of the CCX CKR, such as binding to a chemokine (e.g., ELC, SLC, TECK, BLC or vMIPII). In one embodiment, the polypeptide binds ELC, SLC, and TECK with high affinity.
In a related aspect, the invention provides an isolated polynucleotide that encodes, or is complementary to a sequence that encodes, the CCX CKR polypeptide. In some embodiments the polynucleotide has at least 10, 15, 25, 50 or 100 contiguous bases identical or exactly complementary to SEQ ID NO:1. In various embodiments, the polynucleotide is the full-length sequence of SEQ ID NO:1, encodes a CCX CKR polypeptide of the invention (e.g., having the sequence of SEQ ID NO:2 or a fragment thereof), or selectively hybridizes under high stringent hybridization conditions to a polynucleotide sequence of SEQ ID NO:1. The polynucleotide of the invention may be operably linked to a promoter. The invention provides recombinant vector (e.g., an expression vector) expressing the CCX CKR polypeptides of the invention. In one aspect, the invention provides a polynucleotide having sequence encoding a polypeptide that has an activity (e.g., a chemokine binding activity) of a CCX CKR polypeptide and which is (a) a polynucleotide having the sequence of SEQ ID NO:1 or SEQ ID NO:3; or (b) a polynucleotide which hybridizes under stringent conditions to (a); or (c) a polynucleotide sequence which is degenerate as a result of the genetic code to the sequences defined in (a) or (b).
The invention further provides a cell (e.g., a bacterial, eukaryotic, mammalian, or human cell) containing a recombinant CCX CKR polynucleotide of the invention, and provides a method for producing an CCX CKR protein, peptide, or fusion protein by culturing a cell containing the recombinant CCX CKR polynucleotide under conditions in which the polypeptide is expressed.
In another embodiment, the invention provides an antibody, or antibody fragment, or binding fragment (e.g., produced by phage display) that specifically binds to the CCX CKR polypeptide of the invention. The antibody may be monoclonal and may bind with an affinity of at least about 108/Mxe2x88x921. The invention also provides an isolated cell or a hybridoma capable of secreting the antibody. The antibody may be human or humanized.
In one aspect the invention provides a method of detecting an CCX CKR gene product in a sample by (a) contacting the sample with a probe that specifically binds the gene product, wherein the probe and the gene product form a complex, and detecting the formation of the complex; or (b) specifically amplifying the gene product in the biological sample, wherein said gene product is a polynucleotide, and detecting the amplification product; wherein the formation of the complex or presence of the amplification product is correlated with the presence of the CCX CKR gene product in the biological sample. In one embodiment the gene product is a polypeptide and probe is an antibody. In a different embodiment, the gene product is an RNA and the probe is a polynucleotide.
The invention also provides a method for determining whether a compound does or does not interact directly with the CCX CKR polypeptide, by contacting a chemokine and the CCX CKR polypeptide or ligand binding fragment thereof, adding a test compound, and measuring any decrease in the binding of the chemokine. In various embodiments, the chemokine is ELC, SLC, TECK, BLC, mCTACK, mMIP-1xcex3 or vMIPII or another naturally occurring ligand bound by the CCX CKR. In some embodiments, the chemokine is radiolabeled. Thus, in one aspect, the invention provides a method for identifying a modulator of the binding of CCX CKR to a chemokine by (a) contacting a polypeptide of encoding CCX CKR and the chemokine in the presence of a test compound, and (b) comparing the level of binding of the chemokine and the polypeptide in (a) with the level of binding in the absence of the test compound, wherein a decrease in binding indicates that the test compound is an inhibitor of binding and an increase in binding indicates that the test compound is an enhancer of binding. In one embodiment, the chemokine is ELC, SLC, TECK, BLC, mCTACK, mMIP-1xcex3 or vMIPII. In an embodiment, the CCX CKR polypeptide is expressed by a cell.
In a related aspect, the invention provides a method of identifying a modulator of CCX CKR activity by contacting a cell expressing a recombinant CCX CKR polypeptide and a test compound and assaying for a biological effect that occurs in the presence but not absence of the test compound, wherein a test compound that induces a biological effect is identified as a modulator of CCX CKR activity. In one embodiment, the biological effect assayed for is receptor internalization. In some embodiments, the method also includes the step of contacting the cell with a chemokine that binds the receptor (e.g., ELC, SLC, TECK, BLC, mCTACK, mMIP-1xcex3 or vMIPII) during the assay.
In another related aspect, the invention provides a process for making a pharmaceutical compositon by formulating a modulator of CCX CKR activity (e.g., binding) for pharmaceutical use.
The invention also provides a method for identifying compounds which will be useful for the treatment of CCX CKR-mediated diseases and conditions, by determining whether the compound interacts with the CCX CKR.
In another aspect, the invention provides a method of treating an CCX CKR-mediated condition in a mammal by reducing or increasing the activity or expression of CCX CKR in a cell or tissue in the mammal or administering a modulator of CCX CKR function to the mammal. In various embodiments, the modulator of CCX CKR function is an inhibitor of binding of a chemokine (e.g., ELC) to CCX CKR or an enhancer of binding of a chemokine (e.g., ELC) to CCX CKR. In one embodiment, the invention provides a method of treating a CCX CKR-mediated condition or disease in a subject in need of such treatment by administering an effective amount of a compound that inhibits the binding of the CCX CKR and a chemokine. In various embodiments, the CCX CKR-mediated condition or disease is an inflammatory or allergic disease, an autoimmune disease, graft rejection, cancers, neoplastic diseases, retinopathy, macular degeneration, an infectious disease, or an immunosuppressive disease.